Haynes Lab:Notebook/Engineering PC-TFs/2013/03/25

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Summary

  • Cutting inserts to be placed in mammalian vector

A) Get biobricks and enzymes from freezer.

PstI, SpeI

The following list of parts are:

  • Kozak

Follow steps for gel electrophoresis.

  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Fill gel flask with up to 60 ml of TA buffer.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 40 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
  • Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • Pour gel into tray.
  • Wash the agarose gel flask.
  • Run gel for 45 min at 100 V. Check gel under UV light.
Reagent Volume Stock digest 3/25/2013 Stock digest 3/18/2013
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
SpeI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 3.0 μL
dH2O 5 μL
  30 μL --> 37°C/ ~10 min.


# Biobrick ng/µL
1 Kozak 9.47



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